Characterization of Hsp90 Co-Chaperone p23 Cleavage by Caspase-7 Uncovers a Peptidase–Substrate Interaction Involving Intrinsically Disordered Regions

Abstract

Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, caspase-7 is more proficient at cleaving poly(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure-activity relationship analyses using mutagenesis of the caspase-7 NTD and kinetics show that residues 36-45 of caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD↓G), which is far from the optimal sequence, we tested all residues at that position and found notable differences in the preference of caspase-7 and magnitude of differences between residues compared to the results of studies that have used small peptidic substrate libraries. Finally, bioinformatics shows that the regions we identified in caspase-7 and p23 are intrinsically disordered regions that contain molecular recognition features that permit a transient interaction between these two proteins. In summary, we characterized the binding mode for a caspase that is tailored to the specific recognition and cleavage of a substrate, highlighting the importance of studying the peptidase-substrate pair to understand the modalities of substrate recognition by caspases.

Caspase-9 CARD : core domain interactions require a properly formed active site

“…In caspase-6, an intact prodomain was reported to inhibit self-cleavage at the linker region in vivo (57) and both the prodomain and linker are predicted to be highly disordered protein-binding regions (58) that dramatically affect the stability of caspase-6 (29). In caspase-7 the region just adjacent to the short prodomain contains an exosite for substrate recognition (59,60), so it may be possible that the casp-9 CARD could play similar roles. In the case of caspase-9 it appears that the cleaved state of the intersubunit linker and the interactions between the CARD (prodomain) and the catalytic core is essential for the appropriate function, which is a unique feature of caspase-9.…”

Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis

“…Exosites, which are functional sites distal from the catalytic site, have been shown to play a significant role in the function of caspase-6 and -7 (21,22,38,39). Based on the observation that caspase-3 and -7, which show virtually identical substrate specificity for peptide substrates, utilize an exosite to distinguish native protein substrates (22), we hypothesized that other caspases also utilize exosites to differentiate substrate pools.…”

“…The 42 RRR 44 exosite patch on caspase-6 clearly engages both procaspase-6 and lamin A/C and facilitates their cleavage by caspase-6. The 38 KKKK 41 exosite in caspase-7 engages both PARP and p23 substrates (21,22), and it is possible that additional substrates that engage the site may soon be discovered. To distinguish whether the 42 RRR 44 exosite is used for many substrates or only certain substrates, we analyzed hydrolysis in whole-cell lysates isolated from SK-N-AS neuroblastoma cells.…”

“…Why some substrates are more efficiently recognized by individual caspases and how caspases distinguish their unique pools of substrates is an active area of investigation. The discovery of enzyme exosites (21,22), as well as substrate exosites (39), opens the door to altering caspase activity by targeting either the enzyme or the substrate at an exosite.…”

“…For instance, caspase-7 utilizes an exosite in the N-terminal domain (NTD), 5 a disordered region that lies just after the prodomain, which is proteolytically removed during activation. The patch of caspase-7 consists of a highly positive tetra-lysine 38 KKKK 41 stretch, which helps to recruit protein substrates PARP-1 and p23 (21,22). This exosite enables caspase-7 to cleave these substrates 30-fold more efficiently than caspase-3, despite the fact these two enzymes share the same overall specificity for peptide substrates (23,24).…”